Assessment Report of Planaria (specifically the Flatworm Schmidtea
mediterranea asexual CIW4 clonal line) to amend the Live Import List
Planaria are an important model organism for scientific research and should be added to the live
import list immediately. Though many species of flat worms are readily available in the wild, here in
Australia, the Schmidtea mediterranea species is considered the gold standard planarian model used
in laboratories across the globe. In order to import these important laboratory animals to Australia,
the live import list needs to be amended.
Planaria have the extraordinary ability to regenerate complete individuals from minuscule body
parts. This ability is driven by large numbers of pluripotent stem cells that even in the absence of
injury continuously replace all cell types of the animal. Planarians have therefore become a vital
model system for studying tissue regeneration and stem cell characteristics in the laboratory. Access
to this important model organism will enhance Australia’s standing in biomedical research. Studies
on these animals will build on our current knowledge base in the fields of regenerative medicine and
stem cell biology. Planarians are the model system of choice for addressing the large conceptual
gaps in our current understanding of regeneration, such as how the remaining tissue senses- and
regenerates exactly what’s gone missing or how to activate cell proliferation in adult tissues without
causing cancer. If we can understand these processes in planaria it could lead to the development of
novel therapies to overcome the inherently poor regenerative abilities of humans.
Imported Schmidtea mediterranea of the clonal laboratory strain CIW4 will be housed in a scientific
facility conforming to AQIS guidelines. This will eliminate any potential risk to native planaria
populations.
The following pages address the terms of reference document requirements.
1. Taxonomy of the species:
Like other planarians, Schmidtea mediterranea is a free-living (non-parasitic) freshwater flatworm.
See
figure
1
[3]
(http://en.wikipedia.org/wiki/Schmidtea_mediterranea,
http://genome.wustl.edu/genomes/detail/schmidtea-mediterranea/). The taxonomy of the species
is as follows:
Family:
Dugesiidae
Genus:
Schmidtea (Formerly Dugesia)
Species:
S. mediterranea
Subspecies:
asexual CIW4 clonal line
Taxonomy:
Flatworm
Common name: Planaria
1
A
B
Figure 1: (A) Representative photograph of a S. mediterranea (B) S.
mediterranea physiology.
2. Status of the species under CITES
S. mediterranea are not listed in CITES Appendix I, II or III, nor is it an endangered species.
3. Ecology of the species
S. mediterranea are free-living (non-parasitic) organisms that are a common constituent of fresh
water habitats in their native range (see below). Like other flatworms, they prey on small
arthropods, insects or other small invertebrates. Flatworms themselves are preyed upon by a variety
of predators and therefore occupy intermediate levels of the food chain. Protozoans and other types
of worms parasitize wild flatworm populations, bacteria and fungi can cause infections. No
association of planarians with human or animal pathogens has been described so date.
Lifespan: The clonal CIW4 S. mediterranea asexual strain reproduces by transverse fission and has
been maintained in the laboratory for over 15 years. The animals are thought to be essentially
immortal in the laboratory environment [3]. In the wild, their life span is certainly much shorter,
owing to predation, parasites and infections (see above).
Size and weight: S. mediterranea CIW4 clonal strain can reach sizes of 2 cm in length and 0.4 cm in
width. Like other planarians, S. mediterranea do not have a fixed body size and continuously scale
their body plan proportions within the above size limits [3]. The weight of a single specimen is
therefore highly variable and difficult to assess because of high water content of the body. The
maximal weight remains well below one gram.
Identification of individuals: S. mediterranea CIW4 clonal strain are asexual animals and therefore
do not have sexes. The S. mediterranea CIW4 clonal strain body size is highly variable and it is not
possible to identify individual animals by morphology alone. S. mediterranea can be distinguished
from that of other planarian species by morphology or genetic sequence.
The natural geographic range: There are three reproductive races of S. mediterranea. The egg lying
form lives in a few localities of Corsica, Sardinia, Sicily and one locality in Tunisia. The fissiparous
2
form occurs in Menorca and the city of Barcelona. The third parthenogenetic form lives in a
Sardinian locality. The CIW4 laboratory strain was derived from a single animal of the Barcelona
population.
Habitat: S. mediterranea is found in lowland freshwater environments including spring-fed ponds
and small rivers. They are normally found on hard surfaces such as under rocks and/or gravel
(http://genome.wustl.edu/genomes/detail/schmidtea-mediterranea/). Wild-type S. mediterranea
are found in water with pH ranges between 6.9 and 8.9 and are viable from temperatures above
freezing to about 25C (reduced viability at the upper end of this temperature range). Maximum
salinity detected in Tunisian populations of the species was 2.9 g/l in June.
The Mediterranean climate is a particular variety of subtropical climate that can be found in other
parts of the world including parts of Western and South Australia (See figure 2)
(http://en.wikipedia.org/wiki/Mediterranean_climate)
Figure 2: Parts of the world that replicate the Mediterranean climate
Diet: The diet in the field has not been properly investigated but likely includes small invertebrates
like aquatic snails and mosquito larvae, which are frequently observed to cooccur with S.
mediterranea. The CIW4 laboratory strain also feeds on tubificids (aquatic worms) or Daphnia
(planktonic crustaceans), but is mostly maintained on beef liver for reasons of convenience.
Social behaviour and groupings: S. mediterranea can show grouping behaviour forming
aggregations of individuals under stones. The significance of the grouping behaviour is unknown.
Territorial and aggressive behaviour: S. mediterranea do not show territorial or aggressive
behaviour.
Natural predators: The species is predated by odonate larvae (dragonflies), other large aquatic
invertebrates and fish.
Characteristic that may cause harm to humans and other species: S. mediterranea are free living
non-parasitic worms and are not known to cause harm to humans or other species [3]
3
(http://genome.wustl.edu/genomes/detail/schmidtea-mediterranea/). The CIW4 laboratory strain
has been maintained in laboratories for over 15 years and even the recent DNA and RNA sequencing
efforts have not led to reports of association with any known pathogens.
Reproductive biology: Wild type S. mediterranea worms are hermaphroditic, however the CIW4
laboratory strain derived from a single animal isolated in Barcelona, Spain, only reproduce by
asexual fission. Natural fission involves adhesion of the tail tip to the substrate and pulling away of
the anterior end. The resulting build-up of tension eventually splits the animal in two and both
halves regenerate, thus resulting in two animals. Prior to a subsequent fission event, both fragments
have to grow back to the minimal size required for fission, which in the laboratory environment
requires 2-4 weeks. Fission is stimulated under conditions of low population density, higher
temperatures and occurs predominantly in the dark. Worms can also be cut into pieces and each of
the pieces will form a new complete worm. This is the method of choice for expanding the CIW4
laboratory strain in planarian research labs and the strain has been derived from the clonal
expansion of a single animal.
Age at maturity:
The CIW4 laboratory strain is asexual and never reaches sexual maturity.
Frequency of asexual reproduction: Wild populations of asexual S. mediterranea fission when the
water temperature begins to rise (spring and summer). In winter, the animals generally do not
undergo fission and therefore increase in size, although occasional fission events occur year round.
Female sperm storage:
N/A, since the S. mediterranea CIW4 laboratory strain does not reproduce sexually.
How many eggs or live born are produce with each breeding event:
N/A. Asexual transverse fission results in two worms per fission event.
Hybridisation:
N/A, due to exclusively asexual reproduction strategy.
4. Feral populations.
No feral populations of S. mediterranea CIW4 clonal strain have been reported despite the global
use of the CIW4 laboratory strain as regeneration model. The CIW4 laboratory strain is currently
used in the US, Canada, Japan, France, Spain, Italy, Germany, the UK, India, China, Hong-Kong and
Singapore, amongst others.
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5. Environmental risks
To our knowledge no risk assessment report has been generated discussing S. mediterranea. In fact,
the asexual wild populations of S. mediterranea are in regression, due to human activity and likely
also the inevitable accumulation of deleterious mutations in asexual animals.
6. Likelihood of establishing a breeding population
The S. mediterranea CIW4 clonal laboratory strain will be maintained under laboratory conditions
strictly conforming to AQIS guidelines. A priori, the escape of animals is therefore extremely unlikely.
Ability to find food sources:
Little is known about the food sources of the wild predecessors of the S. mediterranea CIW4
laboratory strain, but it is theoretically possible that similar ecological niches might exist in
Australian fresh water habitats.
Ability to survive and adapt to different climatic conditions:
The current fragmented distribution of the wild predecessors of the CIW4 laboratory strain points to
the fact that asexual S. mediterranea have generally a limited capability to deal with environmental
challenges. Wild S. mediterranea are particularly sensitive to human habitat alterations, as are
prevalent in the vicinity of towns. These effects have led to the extinction of several wild S.
mediterranea populations.
Ability to find shelter:
The dispersion capabilities of the wild predecessors of the CIW4 laboratory strain are limited
because the animals have no desiccation resistant life history stage and a soft, unprotected body.
The animals propel themselves mainly through ciliary movement, which does not allow for effective
long distance migrations. The value of planarians for biogeographical research derives exactly from
their low dispersal capabilities.
Rate of reproduction:
The CIW4 laboratory strain of S. mediterranea can only replicate asexually/clonally as described
above. Under optimal laboratory care conditions (constant temperature, antibiotics, feeding 2x per
week), tail fragments generated by fission will take about 4 weeks to regenerate and grow to the
minimal size limit for fission. Population doubling times can be expected to be significantly longer in
the wild, owing to periodic food supply, temperature fluctuations and infections.
Characteristics enhancing survival:
The S. mediterranea CIW4 clonal laboratory strain does not have any special characteristics that
would enhance its survival in the wild. The clonal origin of the strain and therefore its extremely
limited genetic repertoire can be expected to constitute a major impediment for survival in the wild
and/or the adaptation to new habitats.
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7. Potential Impact of the species
Similar niche species:
There are native Australian planarian species that live in similar niches to S. mediterranea. It is
unlikely that escaped individuals of the CIW4 laboratory strain could outcompete endogenous
species.
Susceptibility to disease/transmission of disease:
The CIW4 laboratory strain of S. mediterranea is not a vector of any known human or animal disease.
Prey/food sources (including agricultural crops):
The wild predecessors of the CIW4 laboratory strain likely prey on small aquatic invertebrates that
are of no commercial value.
Habitat and local environmental conditions:
S. mediterranea is found in lowland freshwater environments including spring-feed ponds and small
rivers. They are normally found at the bottom of hard surfaces such as under rocks and/or gravel.
(http://genome.wustl.edu/genomes/detail/schmidtea-mediterranea/).
Control/eradication programs that could be applied:
Desiccation of the body of water could be a way of eradicating local populations of unwanted
planarian species.
Characteristic or behaviour leading to land degradation:
The wild predecessors of the CIW4 laboratory strain do not exhibit any characteristic or behaviour
that leads to land degradation.
Any potential threats to humans:
The wild predecessors of the CIW4 laboratory strain are free living non-parasitic worms and are not
known to cause harm to humans or other species.
8. Conditions and restrictions:
Import of the CIW4 clonal laboratory strain only.
9. Summary of activities:
Planarians have been used as a laboratory model to study tissue regeneration and plasticity for more
than 200 years [1, 2, 5]. The animals generally have remarkable regenerative capabilities and are
6
able to regenerate complete individuals from miniscule tissue fragments (See figure 2). Exceptionally
abundant pluripotent adult stem cells, so called “neoblasts”, drive the formation of new tissue
during regeneration and the steady turn-over of all planarian cell types in the absence of injury [5].
The recent development of suitable in situ hybridization-, immunocytochemistry, RNAi- and RNAseq
protocols has now rendered the mechanistic basis of regeneration and pluripotent stem cell
regulation accessible to biomedical research.
The planarian species Schmidtea mediterranea is the gold standard laboratory model (see figure 3).
The clonal CIW4 laboratory strain is easy to cultivate under laboratory conditions and harbours a
comparatively small, diploid genome (2n=8, ~7108 bp). The genome of the CIW4 laboratory strain
has been sequenced (http://genome.wustl.edu/genomes/detail/schmidtea-mediterranea/) making
the clonal CIW4 laboratory strain the work horse of planarian research. The clonal CIW4 laboratory
strain is currently the only laboratory model of whole body regeneration or for the homeostatic
control of a pluripotent stem cell system in adult tissues. Findings generated from studies on these
animals are therefore critical to our understanding of stem cell biology and have a strong potential
to be applicable to regenerative medicine and the treatment of human disease
Figure 3: Drawing illustrating some original
regeneration experiments performed in the late
1890s [1, 2] reproduced from [3].
Guidelines for how species should be kept:
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Maintaining worms in laboratory: S. mediterranea will be maintained in the laboratory essentially
using the protocol of Oviedo et al, [6]. Briefly S. mediterranea will be kept in food grade plastic
containers in 1× Montjuïch salts (1.6 mmol/l NaCl, 1.0 mmol/l CaCl2, 1.0 mmol/l MgSO4, 0.1 mmol/l
MgCl2, 0.1 mmol/l KCl and 1.2 mmol/l NaHCO3 prepared in Milli-Q water, pH7.5) and maintained at
20C. Water will be changed every 2-5 days. Animals will be fed weekly with frozen puree of
organic calf liver. The culture will be propagated and expanded via regular rounds of cutting and
regeneration.
Containment/Disposal: This containment procedure is in accordance with the “Post arrival
quarantine requirements for the importation of aquatic animals for laboratory purposes” from AQIS.
1. Animals will remain in the laboratory for the duration of their lives.
2. On arrival, transport containers and all material held therein (excluding the aquatic animals) must
be either: destroyed by autoclave or incineration, or disinfected with 10% bleach. Note a final
concentration of 1.6% sodium hypochlorite (bleach) is equivalent to 200ppm available chlorine.
3. Overseas water, waste water and all water contacting the aquatic animals must be treated prior
to disposal by using a final concentration of 10% sodium hypochlorite (bleach).
4. All parts of the animal’s environment must be immersed in 10% sodium hypochlorite (bleach)
5. All non-disposable equipment within the facility must be disinfected with 10% sodium
hypochlorite (bleach) before being used with other animals.
6. The animals, genetic material and in contact animals are to be used only for research purposes
within the facility.
7. All in-contact animals must remain in the lab and become subject to these conditions.
8. On completion of work, all animals and their progeny and in-contact animals must be euthanised
and disposed of by incineration or autoclaving.
Animals will be contained within secure laboratory premises accessible only by authorised personnel
with an allocated access card.
10. Legislative controls
S. mediterranea do not have a current quarantine status. S. mediterranea is not recognised as a pest
or noxious species anywhere in the world. Nor is this species prohibited or controlled by permit or
licence in any Australian state or territory.
11. Conditions/restrictions
Follow Containment/Disposal procedures as detailed in 10 above.
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References
[1]
[2]
[3]
[4]
[5]
[6]
Randolph H (1897). Observations and experiments on regeneration in planarians. Arch
Entwm Org 5, 352–372.
Morgan TH (1898). Experimental studies of the regeneration of Planaria maculata. Arch
Entwm Org 7, 364–397.
Newmark PA, Sanchez Alvarado A (2002). Not your father's planarian: a classic model enters
the era of functional genomics Nat Rev Genet 3, 210-219.
Benazzi M, Ballester R, Baguñà J, Puccinelli I (1972). The fissiparous race of the planarian
Dugesia lugubris S. L. Found in Barcelona (Spain) belongs to the biotype g: comparative
analysis of the karyotypes. Caryologia 25, 59-68.
Sanchez Alvarado A (2003). The freshwater planarian Schmidtea mediterranea:
embryogenesis, stem cells and regeneration Curr Opin Genet Dev 13, 438-444.
Oviedo NJ, Nicolas CL, Adams DS, Levin M (2008). Establishing and maintaining a colony of
planarians CSH Protoc 2008, pdb prot5053.
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